绢丝昆虫野桑蚕与柳蚕卵黄原蛋白的研究
|
摘要
本论文包括两部分主要内容。第一部分是野桑蚕卵黄原蛋白的鉴定与cDNA序列的分析，以及卵黄组成的研究。利用SDS-PAGE和Western blotting的方法分析鉴定了野桑蚕卵黄原蛋白是由大小两个亚基组成，根据分子量marker得出分子量分别为175 kD和42 kD。对不同性别和不同时期野桑蚕血液的SDS-PAGE分析说明了，野桑蚕卵黄原蛋白存在性别、时期的特异性。通过对野桑蚕吐丝期开始至第5日的雌体脂肪体总RNA的含量进行了比较得出，从吐丝期开始到第5日逐渐递增，与野桑蚕的血液中卵黄原蛋白的含量的变化是一致的。利用昆虫卵黄原蛋白进化上的保守性，根据家蚕的Vg cDNA序列设计4对特异性引物在野桑蚕的总RNA进行RT-PCR扩增，分别得到了4条1000bp左右的cDNA片段：对于3’-end和5’-end进行RACE扩增，分别得到了2条1000bp左右的cDNA片段，根据cDNA片段的重复性，从而解析出野桑蚕卵黄原蛋白cDNA全序列。该序列含有5754碱基，由一个ORF组成，编码1780个氨基酸，卵黄原蛋白的氨基酸序列和cDNA序列与家蚕的同源性分别为97.6％和98.1％。含有特定的酶切识别结构RSRR，卵黄原蛋白前体被酶切为大小两个亚基，根据氨基酸推算的相对分子质量分别为161.571 kD和40.794 kD，如果考虑到翻译后的修饰，这与SDS-PAGE的结果是吻合的。该序列含有一个多聚丝氨酸区，出现在切割位点后的17氨基酸处，DGQR和GICG分别出现在第1581和1597氨基酸处，C末端含有6个保守的半胱氨酸。根据已经解析的18种昆虫卵黄原蛋白的氨基酸序列构建了系统树，结果证明了昆虫卵黄原蛋白的一级结构在进化上具有很好的保守性，尤其是5种绢丝昆虫卵黄原蛋白，它们的氨基酸序列同源性与RAPD分析的结果是一致的。通过家蚕与野桑蚕卵的内容物的SDS-PAGE，分析了野桑蚕卵黄蛋白主要有卵黄磷蛋白(Vtn)，卵雌异性蛋白(ESP)和30K蛋白组成。通过Native-PAGE分析推定了野桑蚕卵黄原蛋白在天然状态下的分子量约为440kD。
第二部分是柳蚕生物学特性以及卵黄原蛋白的鉴定。柳蚕(Actias seleneHübner)是吐丝结茧的经济昆虫之一，属鳞翅目，大蚕蛾科，国内外对其相关的研究较少，作为一种野生绢丝昆虫，对其研究具有很重要的意义。本文通过室内
饲养的方法，观察了柳蚕的生物学特性，在合肥地区幼虫四眠五龄，二化胜，蛹
滞育，全龄期达40d以上。通过扫描电子显微镜观察了柳蚕卵孔的显微特征，
对其形态进行了描述，并与其它野蚕进行了比较。通过SDS一PAGE和Westem
blotting鉴定了柳蚕的卵黄原蛋白是由大小两个亚基组成，分子量分别为1 75 kD
和45 kD，存在组织、时期、性别表达的差异性。
This thesis contains two major parts. The first part is the research of identification of vitellogenin and analysis of its cDNA sequence, and research of composition of yolk protein in Bombyx mandarina Moore. The large subunit and small subunit of vitellogenin in Bombyx mandarina are identified through SDS-PAGE and Western blotting, which molecular weight are about 175kD and 42 kD respectively based on hight molecule weight marker. The vitellogenin in haemolymph have expression difference in different sexes and times. The tendency to change of total RNA in the female fat body of Bombyx mandarina from spinning day 0 to day 5 is consistent with that of vitellogenin in haemolymph of female silkworms. Several primers are designed according to cDNA sequence of vitellogenin in Bombyx mori and conservatism of insect vitellogenins. The cDNA sequence of vitellogenin in Bombyx mandarina is obtained through RT-PCR to the total RNA and RACE amplification to 3'-end and 5'-end, which contains 5754 bases
, an open reading frame which encodes 1780 amino acids, and the putative cleavage sites RSRR for the proteases of the subtilisin family at position 364-367 where provitellogenin is cleaved into two subunits which molecular weight are 161.571kD and 40.794kD respectively based on amino acids, which are in accord with the result of SDS-PAGE if modification is considered after translation. The homology of amino acids and bases between Bombyx mori and Bombyx mandarina are 97.6% and 98.1% respectively. Phylogenetic tree of the entire amino acid sequence of Vg in eighteen insects is constructed and the result proves the conservation among evolution of insect vitellogenins, especially five silk insects and their homology of Vg amino acid is consistent with RAPD analysis. Yolk protens of Bombyx mandarina are also composed of vitellin, egg specific proteins(ESP) and 30K proteins on the base of SDS-PAGE of egg subsracts of Bombyx mori and Bombyx mandarina. Native molecular mass of Vg in the Bombyx mandarina is ar
ound 440kD through Native-PAGE of egg substracts of the two silkworms.
The second part is biology characteristics of Moon silkworm Actias selene
Hiib.ier and identification of its vitellogenin. Moon silkworm Actias selene Hubner is one of economic insects with spinning and cocooning, which belongs to Lepidoptera. There are little study related to moon silkworm at home and abroad. This paper states the biology characteristics of moon silkworm through rearing in room and microcharacters of its egg hole through scanning electron microscope (SEM) , and its instar has four moulting and five stadiums, divoltinism, diapause with pupa and forty days of all instars in Hefei. Two subunits of Vitellogenin in moon silkworm are identified, which molecular weight are about 175kD and 45kD respectively through SDS-PAGE and Western blotting, and also have expression difference in different sexes, tissues, times.
第二部分是柳蚕生物学特性以及卵黄原蛋白的鉴定。柳蚕(Actias seleneHübner)是吐丝结茧的经济昆虫之一，属鳞翅目，大蚕蛾科，国内外对其相关的研究较少，作为一种野生绢丝昆虫，对其研究具有很重要的意义。本文通过室内
饲养的方法，观察了柳蚕的生物学特性，在合肥地区幼虫四眠五龄，二化胜，蛹
滞育，全龄期达40d以上。通过扫描电子显微镜观察了柳蚕卵孔的显微特征，
对其形态进行了描述，并与其它野蚕进行了比较。通过SDS一PAGE和Westem
blotting鉴定了柳蚕的卵黄原蛋白是由大小两个亚基组成，分子量分别为1 75 kD
和45 kD，存在组织、时期、性别表达的差异性。
This thesis contains two major parts. The first part is the research of identification of vitellogenin and analysis of its cDNA sequence, and research of composition of yolk protein in Bombyx mandarina Moore. The large subunit and small subunit of vitellogenin in Bombyx mandarina are identified through SDS-PAGE and Western blotting, which molecular weight are about 175kD and 42 kD respectively based on hight molecule weight marker. The vitellogenin in haemolymph have expression difference in different sexes and times. The tendency to change of total RNA in the female fat body of Bombyx mandarina from spinning day 0 to day 5 is consistent with that of vitellogenin in haemolymph of female silkworms. Several primers are designed according to cDNA sequence of vitellogenin in Bombyx mori and conservatism of insect vitellogenins. The cDNA sequence of vitellogenin in Bombyx mandarina is obtained through RT-PCR to the total RNA and RACE amplification to 3'-end and 5'-end, which contains 5754 bases
, an open reading frame which encodes 1780 amino acids, and the putative cleavage sites RSRR for the proteases of the subtilisin family at position 364-367 where provitellogenin is cleaved into two subunits which molecular weight are 161.571kD and 40.794kD respectively based on amino acids, which are in accord with the result of SDS-PAGE if modification is considered after translation. The homology of amino acids and bases between Bombyx mori and Bombyx mandarina are 97.6% and 98.1% respectively. Phylogenetic tree of the entire amino acid sequence of Vg in eighteen insects is constructed and the result proves the conservation among evolution of insect vitellogenins, especially five silk insects and their homology of Vg amino acid is consistent with RAPD analysis. Yolk protens of Bombyx mandarina are also composed of vitellin, egg specific proteins(ESP) and 30K proteins on the base of SDS-PAGE of egg subsracts of Bombyx mori and Bombyx mandarina. Native molecular mass of Vg in the Bombyx mandarina is ar
ound 440kD through Native-PAGE of egg substracts of the two silkworms.
The second part is biology characteristics of Moon silkworm Actias selene
Hiib.ier and identification of its vitellogenin. Moon silkworm Actias selene Hubner is one of economic insects with spinning and cocooning, which belongs to Lepidoptera. There are little study related to moon silkworm at home and abroad. This paper states the biology characteristics of moon silkworm through rearing in room and microcharacters of its egg hole through scanning electron microscope (SEM) , and its instar has four moulting and five stadiums, divoltinism, diapause with pupa and forty days of all instars in Hefei. Two subunits of Vitellogenin in moon silkworm are identified, which molecular weight are about 175kD and 45kD respectively through SDS-PAGE and Western blotting, and also have expression difference in different sexes, tissues, times.
引文
1. Telfer W H. Immunological studies of insect netamorphosis Ⅱ: The role of a sex-limited blood protein in egg formation by the cecropia silkworm, J. Gen. Physiol., 1954(37): 539-558.
2. Pan M L, Bell W J, Telfer W H. Vitellogenin blood protein synthesis by insect fat body. Science, 1969(165):393-394.
3. Pan M Lo Control of vitellogenin synthesis in the monarch butterfly by juvenile hormone. Dev. Biol., 1977(54): 127-134.
4．李乾君，龚和，管致和．昆虫卵黄发生研究进展．昆虫学报，1995(38)：237-252．
5．李丰倩，黄君霆．昆虫卵黄发生及卵黄原蛋白的研究现状．国外农业—蚕学，1989(3)：12-18．
6. Hagedorn H H, Maddison D R, Tu Z. The evolution of vitellogenins, cyclorrhaphan yolk proteins and related molecules. Adv. Insect Physiol., 1998, 132-139.
7. Byrne, B M, Gruber, M. The evolution of egg yolk proteins. Prog.Biophys.Mol. Biol., 1989(53):33-69.
8. Retzek H, Steyrer E., Sandeers E I, Nimpf J, Schneider, W J. Molecular cloning and functional characterization of chicken cathepsin D, a key enzyme for yolk formation. DNA Cell Biol., 1992(11): 661-672.
9．龚和，翟启慧．昆虫卵黄蛋白和卵黄发生．昆虫学报，1979(22)：219-236．
10. Hiremath S, Lehtoma K. Complete nucleotide sequence of the vitellogenin mRNA from the gypsy moth: novel arrangement of the subunit encoding regions. Insect Biochem.Mol. Biol., 1997 (27) :27-35.
11. Lee J M, Nishimori Y, Hatakeyama M, Bae T W, Oishi K. Vitellogenin of the cicada Graptopsaltria nigrofuscata (Homoptera): analysis of its primary structure. Insect Biochem. Molec. Biol., 2000(30): 1-7.
12. Trewitt P M, Heilmann L J, Degrugillier S S, Kumaran A K. The boll weevil
vitellogenin gene: nucleotide sequence, structure, and evolutionary relationship to nematode and vertebrate vitellogenin genes. J. Mol. Evol., 1992(34): 478-492.
13．王荫长主编．《昆虫生物化学》，中国农业出版社，2000．
14．程家安主编．《昆虫分子科学》，科学出版社，2001．
15. Valle D. Vitellogenesis in insects and other groups—a review. Mem. Inst. Oswaido Cruz., 1993(88): 1-26.
16. Harinsh D G, White B N. An evolutionary model for the insect vitellins. J. Molec. Evol. , 1982(18):405-413.
17. Liu C L, Kajiura Z, Shiomi K, Takei R, Nakagaki M. Purification and cDNA sequencing of vitellogenin of the wild silkworm, Antheraea pernyi. J.Insect. Biotech. Sericul., 2001 (70):95-104.
18. Sappington T W, Railhel A S. Molecular characteristics of insect vitellogenins and vitellogenin receptors. Insect Biochem.Mol. Biol., 1998(28): 277-300.
19. Chen J S, 1997, Sappington T W, Railhel A S. Extensive sequence conservation among insects, nematode, and vertebrate vitellogenins reveal ancient common ancestry. J. Mol. Evol., 1997(44): 440-451.
20. Izumi S, Yano K, Takahashi S Y. Yolk proteins from insect eggs: structure, biosynthesis and programmed degradation during embryogenesis. J Insect Physiol., 1994(40): 735-746.
21. Nose Y, Lee J M, Ueno T, Hatakeyama M, Oishi K. Cloning of cDNA for vitellogenin of the parasitoid wsap, Pimpla nipponica (Hymenopetera: Aprcrita: Ichneumoidae): vitellogenin primary structure and evolutionary considerations. Insect Biochem.Molec.Biol. , 1997(27): 47-1056.
22．刘朝良，梶浦善太，中垣雅雄，武井隆三．柞蚕卵巢移植于雄体后的发育及其卵黄原蛋白的吸收积累．安徽农业大学学报，2000，27(3)：272-274．
23. Liu C L, Kajiura Z, Takei R, Nakagaki M. Reciprocal transplant of an ovarian imaginal disc and uptake of vitellogenin bu the ovaries of two wild silkworm, Antheraea pernyi and Antheraea yamamai. J. Seric. Jpn., 2000(69): 251-260.
24. Piulachs M P, Guidugli K R, Barchuk A R, Cruz J. The vitellogenin of the
honeybee, Apis mellifera: structural analysis of the cDNA and expression studies. Insect Biochem. Molec. Biol., 2003(33): 459-465.
25. Yano K, Sakurai M T, Watabe S, Izumi S, Tomino S. Structure and expression of mRNA for vitellogenin in Bombyx mori. Biochim. Biophys. Acta, 1994(1218): 1-10.
26．叶恭银，胡萃，洪健，龚和．天蚕卵黄原蛋白的合成、运转与沉积．昆虫学报，1999(42)：225-233．
27. Kageyama Y, Kinoshita T, Umesono Y, Hatakeyama M, Oishi K. Cloning of cDNA for vitellogenin of Athalia rosae (Hymenoptera) and characterization of the vitellogenin gene expression, Insect Biochem Mol. Biol., 1994(24): 599-605.
28. Tufail M, Lee J M, Hatakeyama M, Oishi K, Takeda M. Cloning of vitellogenin cDNA of the American co.ckroach, Periplaneta Americana (Dictyoptera) and its structural and expression analysis. Arch. Insect Biochem. Physiol., 2000(45): 37-46.
29. Hirai H, Watanabe D, Kiyota A, Chinzei Y. Nucleotide sequence of vitellogeinin mRNA in the bean bug, Riptortus clavatus: analysis of processing in the fat body and ovary. Insect Biochem. Mol Biol., 1998(28): 537-547.
30. Martin D, Piulachs, M D, Comas D, Belles X. Isolation and sequence of a partial vitellogenin cDNA from the cockroach Blattella germanica (Dictyopetra, Blattellidae), and characterization of the vitellogenin gene expression. Arch Insect Biochem. Physiol., 1998(38): 137-146.
31. Chen T T, Strahlendrof P W, Wyatt G R. Vitellin and vitellogenin from locust (Locusta migratoria): Properties and post-transnational modification in the fat body. J. Biol. Chem., 1978(253): 441-445.
32．陈建新，沈杰，宋敦伦，张龙，严毓华．蝗虫微孢子虫对东亚飞蝗卵黄原蛋白含量的影响，昆虫学报，2002(45)：170-172．
33. Reid P, Chen T T. Juvenile hormone control vitellogenin synthesis in the fat body of the locust (Locusta migratoria): Isolation and characterization of vitellogenin polysome and their induction in vivo. Insect Biochem.,
1981 (11):297-305
34. Wyatt G R, Cook K E. Juvenile hormone action on locust fat body. Insect Biochem., 1987(17): 1071 - 1073.
35．龚和，翟启慧，魏守义，张建中．七星瓢虫卵黄发生的研究：卵黄原蛋白的发生规律及取食代饲料的影响．昆虫学报，1980(23)：252-259．
36．龚和，张建中，翟启慧．七星瓢虫卵黄发生的合成．动物学集刊，1982(2)：175-181．
37. Zhai Q H, Zhang J Z, Gong H. Regulation of vitellogenin synthesis by juvenile hormone analogue in Cocinella septempunctata. Insect Biochem., 1987(17): 1064-1095.
38. Zhai Q H, Postlethwait J H, Bodley J W. Vitellogenin synthesis in the lady bettle Cocinella septempunctata. Insect Biochem., 1984(14):299-305.
39．翟启慧，张建中．七星瓢虫的卵黄发生：体外培养脂肪体中卵黄蛋白的合成．昆虫学报，1985(28)：211-218．
40．翟启慧，龚和，王成，郑文惠．七星瓢虫卵黄原蛋白基因的表达：保幼激素类似物对mRNA的诱导．昆虫学报，1990(33)：257-264．
41．于长明，刘泉，李成文．保幼激素类似物对德国小蠊卵黄发生及繁殖的影响．昆虫学报，1999(42)：353-357．
42．龚和，李乾君．家蝇的卵黄发生及其激素调节．昆虫学报，1992(35)：129-137．
43．李乾君，管致和，龚和．早熟素Ⅱ号对家蝇卵黄发生的影响．昆虫学报，1993(36)：129-137．
44. Dhadialla T S, Hays A R, Raikhel A S. Characterization of the solubilized mosquito vitellogenin receptor. Insect Biochem. Molec. Biol. 1992(22): 803-816.
45. Osir E O, Lav J H. Studies on binding and uptake of vitellogenin by follicles of the tobacce homworm, Manduca Sexta. Arch. Insect Biochem. Physiol., 1986(3): 523-528.
46．龚和，邱威，郑文惠，李乾君．家蝇卵巢摄取卵黄蛋白的机理．昆虫学报，
1994(37):8-15.
47. Davis R E, Kelly T J, Masler E P, Fescemyer H W, Thyagaraja B S, Borkovec A B. Hormonal control of vitellogenesis in the gypsy month, Lymantria dispar (L): Suppression of haemolymph vitellogenin by the juvenile hormone analogue methoprene. J Insect Physiol., 1990(36): 231-238.
48．吴小锋，徐俊良．蜕皮激素对家蚕卵黄蛋白合成积累及卵巢发育的影响．蚕业科学，1992(1 8)：20-24．
49. Goldstein J L, Anderson R G W, Brown M S. Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature, 1979(279): 679-685.
50. Davis C G, van Driel I R, Russell D W, Brown M S, Goldstein J L. The low-density lipoprotein receptor: identification of amino acids in the cytoplasmic domain required for rapid endocytosis. J. Biol. Chem., 1987(262): 4075 -4082.
51. Davis C G, Goldstein J L, Sudhof T C, Russell D W, Brown M S. Acid-dependent ligand dissociation and recycling of LDL receptor mediated by growth factor homology region. Nature, 1987(326): 760-765.
52. Sappington T W, Hays A R, Railhel A S. Mosquito vitellogenin receptor: purification, development and biochemical characterization, Insect Biochem. Molec. Biol. 1995(25): 807-817.
53. Fox J W, Mayer U, Nischt R, Aumailley M, Wiodemann H. Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type Ⅳ. EMBOJ,, 1991(10): 3127-3146.
54. Mayer U, Nischt R, Mann K, Gerl M. A single EGF-like motif of laminin is responsible for high affinity nidogen binding. EMBOJ, 1993(12): 1879-1885.
55．桂慕燕，左正宏，陈元霖．五种绢丝昆虫随机扩增多态性DNA分析．遗传，2001(23)：25-28．
56．Yamashita，Nature，1980(283)：385-386．
57．朱江，戴玉锦．家蚕卵黄蛋白质分子生物学研究进展．国外农学蚕业，1989(3)：4-11．
58. Hagedorn H H, Kunkel J G. Vitellogenin and vitellin in insects. Ann. Rew. Entomol. , 1979(24):475-505.
59. Brownes B, 1986. Expression of the gene coding for vitellogenin (yolk protein). Ann. Rew. Entomol., 1986(31):507-531.
60．董胜张，刘朝良，汪泰初，朱保健．绢丝昆虫柳蚕的生物学特性和卵黄原蛋白鉴定．蚕业科学，2003(4)：439-442．
61. Narumi Yoshitake. Phylogenetic aspects on the origin of Japanese race of the silkworm, Bombyx mori L. J. Seric. Sci. Jpn., 1967 (37) :83-87.
62．蒋猷龙．《家蚕的起源和分化》，南京：江苏科技出版社，1982．
63．李焕文．野桑蚕与家蚕的起源及其化性变化的探索．蚕学通讯，1985(1)：42-45．
64．于继彬，季平．野桑蚕的遗传多样性及其研究进展．中国蚕业，2001(4)：51-52．
65．彭卫平．家蚕和中国野桑蚕远缘杂交育种的初步研究．安徽农业大学学报，1994(21)：172-176．
66．徐厚镕，金长江，董胜张．He-Ne激光辐照野桑蚕子代变异体继代遗传的研究．蚕桑茶叶通讯，2002(1)：3-5．
67．金长江，徐厚镕，陈复生．He-Ne激光辐照野桑蚕生物学效应的研究．激光生物学报，2001(10)：96-100．
68．鲁成，赵爱春，向仲怀，万春玲．中国野桑蚕遗传多样性的AFLP分析．动物学研究，2002(23)：166-172．
69．鲁成，余红仕，向仲怀．基于RAPD分析的中国野桑蚕和家蚕遗传多样性和系统发育关系的研究．昆虫学报，2002(45)：198-203．
70．郭尧君主编．《蛋白质电泳实验技术》．科学出版社，1999．
71．(美)J．萨姆布鲁克等．《分子克隆实验指南》第3版．北京：科学出版社，1996．
72．(美)J．萨姆布鲁克等．《分子克隆实验指南》第2版．北京：科学出版社，1996．
73. Zenta K, Takesi O, Masao N, Ryuzo T. Female accessory glands of the wild
silkmothes and their secretory proteins. J. Seric. Sci. Jpn., 1997(66) : 413-419.
74. Hiremath S. Lehtoma K. Complete nucleotide sequence of the vitellogenin mRNA from the gypsy moth: novel arrangement of the subunit encoding regions. Insect Biochem.Mol. Biol., 1997(27): 27-35.
75. Chen J S, Raikhel A S. Subunit cleavage of mosquito provitellogenin by a subtilisim-like convertase. Proc. Natl. Sci. Acad USA, 1996(93): 6186-6190.
76. Mouchel N, Trichet V, Betz A, Pennec J-P L, Wolff J. Characterization of vitellogenin from rainbow trout (Oncorhynchus mykiss). Gene, 1996(174): 59-64.
77．刘朝良，吴洁梅，梶浦善太．柞蚕脂肪体RNA量的消长与体液卵黄原蛋白含量的关系．蚕业科学，2003(28)：199-202．
78. Comas D, Piulachs, M-D, Belles, X. Vitellogenin of Blattella germanica (L.)(Dictyoptera, Blattellidae): Nucleotide sequence of the cDNA and analysis of the protein primary structure, lnsect Bioch.Physio., 2000(45): 1-11.
79．刘朝良，梶浦善太，中垣雅雄．蓖麻蚕卵黄原蛋白cDNA的克隆及序列分析．激光生物学报，2003(12)：12-17．
80．夏庆友，周泽汤，鲁成，向仲怀．家蚕不同地理品种(系)分子系统学研究．昆虫学报，1998(41)：32-40．
81．左正宏，桂幕燕，陈元霖，王学民．RAPD分析在绢丝昆虫亲缘关系研究中的应用．遗传．2001(23)：128-130．
82．张春玲，陈元霖，桂幕燕，刘春宇．蓖麻蚕DNA导入引起家蚕遗传变异的研究——基因组DNA的RAPD检测．遗传，1998(20)：1-4．
83．苏伦安主编．《野蚕学》．北京：中国农业出版社，1991：272～273．
84．宋方洲，蒋同庆．绢丝昆虫柳天蚕(Actias seleneHübner)的形态习性．蚕学通讯，1989(4)：14-17．
85．胡萃主编．《天蚕研究论文集》．上海科学技术出版社，1990：8-9．
2. Pan M L, Bell W J, Telfer W H. Vitellogenin blood protein synthesis by insect fat body. Science, 1969(165):393-394.
3. Pan M Lo Control of vitellogenin synthesis in the monarch butterfly by juvenile hormone. Dev. Biol., 1977(54): 127-134.
4．李乾君，龚和，管致和．昆虫卵黄发生研究进展．昆虫学报，1995(38)：237-252．
5．李丰倩，黄君霆．昆虫卵黄发生及卵黄原蛋白的研究现状．国外农业—蚕学，1989(3)：12-18．
6. Hagedorn H H, Maddison D R, Tu Z. The evolution of vitellogenins, cyclorrhaphan yolk proteins and related molecules. Adv. Insect Physiol., 1998, 132-139.
7. Byrne, B M, Gruber, M. The evolution of egg yolk proteins. Prog.Biophys.Mol. Biol., 1989(53):33-69.
8. Retzek H, Steyrer E., Sandeers E I, Nimpf J, Schneider, W J. Molecular cloning and functional characterization of chicken cathepsin D, a key enzyme for yolk formation. DNA Cell Biol., 1992(11): 661-672.
9．龚和，翟启慧．昆虫卵黄蛋白和卵黄发生．昆虫学报，1979(22)：219-236．
10. Hiremath S, Lehtoma K. Complete nucleotide sequence of the vitellogenin mRNA from the gypsy moth: novel arrangement of the subunit encoding regions. Insect Biochem.Mol. Biol., 1997 (27) :27-35.
11. Lee J M, Nishimori Y, Hatakeyama M, Bae T W, Oishi K. Vitellogenin of the cicada Graptopsaltria nigrofuscata (Homoptera): analysis of its primary structure. Insect Biochem. Molec. Biol., 2000(30): 1-7.
12. Trewitt P M, Heilmann L J, Degrugillier S S, Kumaran A K. The boll weevil
vitellogenin gene: nucleotide sequence, structure, and evolutionary relationship to nematode and vertebrate vitellogenin genes. J. Mol. Evol., 1992(34): 478-492.
13．王荫长主编．《昆虫生物化学》，中国农业出版社，2000．
14．程家安主编．《昆虫分子科学》，科学出版社，2001．
15. Valle D. Vitellogenesis in insects and other groups—a review. Mem. Inst. Oswaido Cruz., 1993(88): 1-26.
16. Harinsh D G, White B N. An evolutionary model for the insect vitellins. J. Molec. Evol. , 1982(18):405-413.
17. Liu C L, Kajiura Z, Shiomi K, Takei R, Nakagaki M. Purification and cDNA sequencing of vitellogenin of the wild silkworm, Antheraea pernyi. J.Insect. Biotech. Sericul., 2001 (70):95-104.
18. Sappington T W, Railhel A S. Molecular characteristics of insect vitellogenins and vitellogenin receptors. Insect Biochem.Mol. Biol., 1998(28): 277-300.
19. Chen J S, 1997, Sappington T W, Railhel A S. Extensive sequence conservation among insects, nematode, and vertebrate vitellogenins reveal ancient common ancestry. J. Mol. Evol., 1997(44): 440-451.
20. Izumi S, Yano K, Takahashi S Y. Yolk proteins from insect eggs: structure, biosynthesis and programmed degradation during embryogenesis. J Insect Physiol., 1994(40): 735-746.
21. Nose Y, Lee J M, Ueno T, Hatakeyama M, Oishi K. Cloning of cDNA for vitellogenin of the parasitoid wsap, Pimpla nipponica (Hymenopetera: Aprcrita: Ichneumoidae): vitellogenin primary structure and evolutionary considerations. Insect Biochem.Molec.Biol. , 1997(27): 47-1056.
22．刘朝良，梶浦善太，中垣雅雄，武井隆三．柞蚕卵巢移植于雄体后的发育及其卵黄原蛋白的吸收积累．安徽农业大学学报，2000，27(3)：272-274．
23. Liu C L, Kajiura Z, Takei R, Nakagaki M. Reciprocal transplant of an ovarian imaginal disc and uptake of vitellogenin bu the ovaries of two wild silkworm, Antheraea pernyi and Antheraea yamamai. J. Seric. Jpn., 2000(69): 251-260.
24. Piulachs M P, Guidugli K R, Barchuk A R, Cruz J. The vitellogenin of the
honeybee, Apis mellifera: structural analysis of the cDNA and expression studies. Insect Biochem. Molec. Biol., 2003(33): 459-465.
25. Yano K, Sakurai M T, Watabe S, Izumi S, Tomino S. Structure and expression of mRNA for vitellogenin in Bombyx mori. Biochim. Biophys. Acta, 1994(1218): 1-10.
26．叶恭银，胡萃，洪健，龚和．天蚕卵黄原蛋白的合成、运转与沉积．昆虫学报，1999(42)：225-233．
27. Kageyama Y, Kinoshita T, Umesono Y, Hatakeyama M, Oishi K. Cloning of cDNA for vitellogenin of Athalia rosae (Hymenoptera) and characterization of the vitellogenin gene expression, Insect Biochem Mol. Biol., 1994(24): 599-605.
28. Tufail M, Lee J M, Hatakeyama M, Oishi K, Takeda M. Cloning of vitellogenin cDNA of the American co.ckroach, Periplaneta Americana (Dictyoptera) and its structural and expression analysis. Arch. Insect Biochem. Physiol., 2000(45): 37-46.
29. Hirai H, Watanabe D, Kiyota A, Chinzei Y. Nucleotide sequence of vitellogeinin mRNA in the bean bug, Riptortus clavatus: analysis of processing in the fat body and ovary. Insect Biochem. Mol Biol., 1998(28): 537-547.
30. Martin D, Piulachs, M D, Comas D, Belles X. Isolation and sequence of a partial vitellogenin cDNA from the cockroach Blattella germanica (Dictyopetra, Blattellidae), and characterization of the vitellogenin gene expression. Arch Insect Biochem. Physiol., 1998(38): 137-146.
31. Chen T T, Strahlendrof P W, Wyatt G R. Vitellin and vitellogenin from locust (Locusta migratoria): Properties and post-transnational modification in the fat body. J. Biol. Chem., 1978(253): 441-445.
32．陈建新，沈杰，宋敦伦，张龙，严毓华．蝗虫微孢子虫对东亚飞蝗卵黄原蛋白含量的影响，昆虫学报，2002(45)：170-172．
33. Reid P, Chen T T. Juvenile hormone control vitellogenin synthesis in the fat body of the locust (Locusta migratoria): Isolation and characterization of vitellogenin polysome and their induction in vivo. Insect Biochem.,
1981 (11):297-305
34. Wyatt G R, Cook K E. Juvenile hormone action on locust fat body. Insect Biochem., 1987(17): 1071 - 1073.
35．龚和，翟启慧，魏守义，张建中．七星瓢虫卵黄发生的研究：卵黄原蛋白的发生规律及取食代饲料的影响．昆虫学报，1980(23)：252-259．
36．龚和，张建中，翟启慧．七星瓢虫卵黄发生的合成．动物学集刊，1982(2)：175-181．
37. Zhai Q H, Zhang J Z, Gong H. Regulation of vitellogenin synthesis by juvenile hormone analogue in Cocinella septempunctata. Insect Biochem., 1987(17): 1064-1095.
38. Zhai Q H, Postlethwait J H, Bodley J W. Vitellogenin synthesis in the lady bettle Cocinella septempunctata. Insect Biochem., 1984(14):299-305.
39．翟启慧，张建中．七星瓢虫的卵黄发生：体外培养脂肪体中卵黄蛋白的合成．昆虫学报，1985(28)：211-218．
40．翟启慧，龚和，王成，郑文惠．七星瓢虫卵黄原蛋白基因的表达：保幼激素类似物对mRNA的诱导．昆虫学报，1990(33)：257-264．
41．于长明，刘泉，李成文．保幼激素类似物对德国小蠊卵黄发生及繁殖的影响．昆虫学报，1999(42)：353-357．
42．龚和，李乾君．家蝇的卵黄发生及其激素调节．昆虫学报，1992(35)：129-137．
43．李乾君，管致和，龚和．早熟素Ⅱ号对家蝇卵黄发生的影响．昆虫学报，1993(36)：129-137．
44. Dhadialla T S, Hays A R, Raikhel A S. Characterization of the solubilized mosquito vitellogenin receptor. Insect Biochem. Molec. Biol. 1992(22): 803-816.
45. Osir E O, Lav J H. Studies on binding and uptake of vitellogenin by follicles of the tobacce homworm, Manduca Sexta. Arch. Insect Biochem. Physiol., 1986(3): 523-528.
46．龚和，邱威，郑文惠，李乾君．家蝇卵巢摄取卵黄蛋白的机理．昆虫学报，
1994(37):8-15.
47. Davis R E, Kelly T J, Masler E P, Fescemyer H W, Thyagaraja B S, Borkovec A B. Hormonal control of vitellogenesis in the gypsy month, Lymantria dispar (L): Suppression of haemolymph vitellogenin by the juvenile hormone analogue methoprene. J Insect Physiol., 1990(36): 231-238.
48．吴小锋，徐俊良．蜕皮激素对家蚕卵黄蛋白合成积累及卵巢发育的影响．蚕业科学，1992(1 8)：20-24．
49. Goldstein J L, Anderson R G W, Brown M S. Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature, 1979(279): 679-685.
50. Davis C G, van Driel I R, Russell D W, Brown M S, Goldstein J L. The low-density lipoprotein receptor: identification of amino acids in the cytoplasmic domain required for rapid endocytosis. J. Biol. Chem., 1987(262): 4075 -4082.
51. Davis C G, Goldstein J L, Sudhof T C, Russell D W, Brown M S. Acid-dependent ligand dissociation and recycling of LDL receptor mediated by growth factor homology region. Nature, 1987(326): 760-765.
52. Sappington T W, Hays A R, Railhel A S. Mosquito vitellogenin receptor: purification, development and biochemical characterization, Insect Biochem. Molec. Biol. 1995(25): 807-817.
53. Fox J W, Mayer U, Nischt R, Aumailley M, Wiodemann H. Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type Ⅳ. EMBOJ,, 1991(10): 3127-3146.
54. Mayer U, Nischt R, Mann K, Gerl M. A single EGF-like motif of laminin is responsible for high affinity nidogen binding. EMBOJ, 1993(12): 1879-1885.
55．桂慕燕，左正宏，陈元霖．五种绢丝昆虫随机扩增多态性DNA分析．遗传，2001(23)：25-28．
56．Yamashita，Nature，1980(283)：385-386．
57．朱江，戴玉锦．家蚕卵黄蛋白质分子生物学研究进展．国外农学蚕业，1989(3)：4-11．
58. Hagedorn H H, Kunkel J G. Vitellogenin and vitellin in insects. Ann. Rew. Entomol. , 1979(24):475-505.
59. Brownes B, 1986. Expression of the gene coding for vitellogenin (yolk protein). Ann. Rew. Entomol., 1986(31):507-531.
60．董胜张，刘朝良，汪泰初，朱保健．绢丝昆虫柳蚕的生物学特性和卵黄原蛋白鉴定．蚕业科学，2003(4)：439-442．
61. Narumi Yoshitake. Phylogenetic aspects on the origin of Japanese race of the silkworm, Bombyx mori L. J. Seric. Sci. Jpn., 1967 (37) :83-87.
62．蒋猷龙．《家蚕的起源和分化》，南京：江苏科技出版社，1982．
63．李焕文．野桑蚕与家蚕的起源及其化性变化的探索．蚕学通讯，1985(1)：42-45．
64．于继彬，季平．野桑蚕的遗传多样性及其研究进展．中国蚕业，2001(4)：51-52．
65．彭卫平．家蚕和中国野桑蚕远缘杂交育种的初步研究．安徽农业大学学报，1994(21)：172-176．
66．徐厚镕，金长江，董胜张．He-Ne激光辐照野桑蚕子代变异体继代遗传的研究．蚕桑茶叶通讯，2002(1)：3-5．
67．金长江，徐厚镕，陈复生．He-Ne激光辐照野桑蚕生物学效应的研究．激光生物学报，2001(10)：96-100．
68．鲁成，赵爱春，向仲怀，万春玲．中国野桑蚕遗传多样性的AFLP分析．动物学研究，2002(23)：166-172．
69．鲁成，余红仕，向仲怀．基于RAPD分析的中国野桑蚕和家蚕遗传多样性和系统发育关系的研究．昆虫学报，2002(45)：198-203．
70．郭尧君主编．《蛋白质电泳实验技术》．科学出版社，1999．
71．(美)J．萨姆布鲁克等．《分子克隆实验指南》第3版．北京：科学出版社，1996．
72．(美)J．萨姆布鲁克等．《分子克隆实验指南》第2版．北京：科学出版社，1996．
73. Zenta K, Takesi O, Masao N, Ryuzo T. Female accessory glands of the wild
silkmothes and their secretory proteins. J. Seric. Sci. Jpn., 1997(66) : 413-419.
74. Hiremath S. Lehtoma K. Complete nucleotide sequence of the vitellogenin mRNA from the gypsy moth: novel arrangement of the subunit encoding regions. Insect Biochem.Mol. Biol., 1997(27): 27-35.
75. Chen J S, Raikhel A S. Subunit cleavage of mosquito provitellogenin by a subtilisim-like convertase. Proc. Natl. Sci. Acad USA, 1996(93): 6186-6190.
76. Mouchel N, Trichet V, Betz A, Pennec J-P L, Wolff J. Characterization of vitellogenin from rainbow trout (Oncorhynchus mykiss). Gene, 1996(174): 59-64.
77．刘朝良，吴洁梅，梶浦善太．柞蚕脂肪体RNA量的消长与体液卵黄原蛋白含量的关系．蚕业科学，2003(28)：199-202．
78. Comas D, Piulachs, M-D, Belles, X. Vitellogenin of Blattella germanica (L.)(Dictyoptera, Blattellidae): Nucleotide sequence of the cDNA and analysis of the protein primary structure, lnsect Bioch.Physio., 2000(45): 1-11.
79．刘朝良，梶浦善太，中垣雅雄．蓖麻蚕卵黄原蛋白cDNA的克隆及序列分析．激光生物学报，2003(12)：12-17．
80．夏庆友，周泽汤，鲁成，向仲怀．家蚕不同地理品种(系)分子系统学研究．昆虫学报，1998(41)：32-40．
81．左正宏，桂幕燕，陈元霖，王学民．RAPD分析在绢丝昆虫亲缘关系研究中的应用．遗传．2001(23)：128-130．
82．张春玲，陈元霖，桂幕燕，刘春宇．蓖麻蚕DNA导入引起家蚕遗传变异的研究——基因组DNA的RAPD检测．遗传，1998(20)：1-4．
83．苏伦安主编．《野蚕学》．北京：中国农业出版社，1991：272～273．
84．宋方洲，蒋同庆．绢丝昆虫柳天蚕(Actias seleneHübner)的形态习性．蚕学通讯，1989(4)：14-17．
85．胡萃主编．《天蚕研究论文集》．上海科学技术出版社，1990：8-9．